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Abstract
As a strategy to understand cellular mechanisms of drought tolerance, we analyzed the electrophoretic pattern of
cell wall proteins from non adapted cells (ST) and a 15% polyethylenglycol (PEG)-tolerant clone (T7) of chili pepper
(Capsicum annuum). To separate proteins bound to structural polymers by non-covalent links or disulfide bonds, cell
walls were treated with sodium dodecyl sulphate (SDS), urea and 2-mercaptoethanol. After treatment three major
proteins with apparent molecular masses of 9, 11 and 14 kDa accumulated in higher quantities in clone T7 than in
ST cells. Cell walls were also solubilized with commercial lytic enzymes, such as lyticase and cellulase, to separate
proteins joined by covalent bonds. The main difference was the presence of a new band with an apparent molecular
mass of 10 kDa (p10) in clone T7 but not in ST cells. This band was also evident after autodigestion of walls by
hydrolytic enzymes. Since p10 was liberated by cellulase and lyticase we suggest that this protein is covalently
attached to the cellulose and glucan skeleton in clone T7. The potential role of p10 on the mechanism of drought
tolerance is discussed. 1997 Elsevier Science Ireland Ltd.
Keywords: Capsicum annuum; Cell wall proteins; Drought tolerance
1. Introduction
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3. Results
To determine whether changes in the cell wall
structure are related to the mechanisms of adaptation to water stress, we analyzed the composition
of cell wall proteins from cellular suspensions of
chili pepper with different tolerances to PEG.
Proteins associated by non-covalent links were
separated by treatment of the cell walls with SDS,
urea or 2-mercaptoethanol. In the electrophoretic
analysis of SDS-solubilized cell wall proteins from
ST cells it was not possible to distinguish individual bands (data not shown). Therefore, we used
15 mg of dry cell walls and the solubilized material was analyzed. The results showed a pattern of
bands with apparent molecular masses in the
range of 752 kDa (Fig. 1, lane a). By using the
same amount of clone T7 derived-walls, we didnt
obtain a good resolution of low molecular weigth
bands, since an over loading of the gel was evident (Fig. 1, lane b); a higher expression of three
proteins with apparent molecular masses between
40 and 52 kDa was observed. The solubilization
of 5 mg dry lyophilized walls from clone T7
showed five major bands with apparent molecular
masses of 9, 11, 14, 16 and 20 (referred to as p9,
p11, p14, p16 and p20, respectively); these bands
appeared with higher intensity in clone T7 than in
extracts of ST cells (Fig. 1, lane c).
After treatment with urea, p9, p11, p12, p14
and p20 were particularly distinguished in ST
cells. A substantial increase in p9, p11 and p14
was seen in clone T7 (Fig. 2A). Cell walls solubilized with 2-mercaptoethanol also showed differences between ST and clone T7 cells (Fig. 2B).
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4. Discussion
Cell walls in higher plants are made up of
complexes of cellulosic microfibrils and a non-cellulosic matrix composed of pectins, hemicelluloses
and proteins [17]. The synthesis and the crosslinking of these wall proteins are under strict control
and can be environmentally induced by a number
of factors. For example, there is evidence supporting the existence of an extensin-pectin crosslinking
[8]. The positively charged lysine and protonated
histidine residues of extensin could interact with
the negatively charged uronic acid of pectins.
Such interactions can be regulated by changes in
cell wall pH and Ca2 + and thus alter the physicochemical properties of the wall. The expression of
glycine-rich proteins is also regulated by a variety
of stress conditions, including ABA and drought
stress [8].
On the other hand, tolerance to water stress is
related to a higher elasticity of cell wall, described
by the so-called volumetric elastic modulus (o).
Low o provide cells with a higher resistance to
short-term fluctuations in the cw of the environment. A high o value insures that relative changes
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Autolysis of cell wall during in vitro incubation is due to glycosylases associated to this
structure. The results corroborated that p10
specifically accumulated in clone T7 but not in
ST cells. The electrophoretic analysis also revealed the presence of p9 and p34, probably
solubilized when the fibrillar structure of cell
wall of T7 was totally digested after the long
period of incubation with its own lytic enzymes
(Fig. 4).
Since clone T7 has been mantained for several years in 15% PEG it is difficult to believe
that appearance of p10 was the result of cellular damage or just a consequence of water
stress. The possibility that some of the differences between ST cells an clone T7 was due to
contaminating proteases is low, since cell walls
were extracted at 4C in the presence of PMSF.
Besides the electrophoretic pattern obtained after 24 h of incubation (Fig. 4) was very similar
to that obtained at 5 min (Fig. 1a).
We believe that the precise localization and
spatial orientation of the fibrilar and amorphous components of cell wall are implicated
directly in its final architecture and function.
The incorporation of p10 may participate in the
supramolecular arrangement of the cell wall, either as an enzyme or as a structural protein,
acting as scaffolds for the deposition of other
molecules or changing the cell-wall-plasma
membrane continuum; p10 probably could also
affect the mechanical strength of the cell wall
by increasing the value of o and consequently
reducing the changes in cell water content.
Further studies are required to elucidate the
distribution and topology of p10 in cell wall, as
well as its contribution to mechanism of tolerance to water deficit in clone T7. The use of
specific antibodies may permit us to determine
the localization of p10 at the ultrastructural
level. Another strategy to study the role of p10
in the mechanism of tolerance to PEG, would
be to correlate its presence with water stress. If
p10 were an inducible protein, we would expect
its disappearance from the cell wall after gradually transfering clone T7 to media without
PEG.
Acknowledgements
We are indebted to Dr Everardo Lopez
Romero for critical review of the manuscript.
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